We intend to extend genomics from the characterization of individual bacterial genomes to characterizing the evolution of infectious disease-causing bacteria. The Chlamydiales, an order of Gram-negative obligate intracellular pathogens, are ideally suited to this approach. Members of this order cause a range of diseases but continue to be refractory to conventional genetic analyses. Our novel, multifaceted approach, which we have named "Taxogenomics", is to first sequence the type strains of the Chlamydiaceae species not addressed by previous or current projects - C. suis, C. pecorum, and C. psittaci (a potential biological warfare agent). Additionally, we will sequence the koala strain of C. pneumoniae and the most evolutionarily distant members of the Chlamydiales, Waddlia chondrophila and Simkania negevensis. To identify those genes which may have been lost by the laboratory adapted type strains or been acquired by "wild" isolates, we will apply subtractive hybridization by pooling multiple isolates from each species and subtracting those genes shared by the type strain. With sequences from representatives of all genomes and with sequences derived from the taxonomic genomic subtractions, we will then create a gene database of all known ORFs from this evolutionarily isolated family. We will use this database to design a non-redundant microarray of Chlamydiales gene variation (called here the "Taxochip"). In this study, we will use the Taxochip in a suite of comparative genomic hybridization (CGH) experiments using a unique set of diverse Chlamydiales isolates that we have compiled from around the globe. We expect to use this genome data to investigate the evolution of the Chlamydiales and identify those genes that may differentiate the variety of observed host/tissue niches and resulting disease outcomes. We also will assess the potentially important role of horizontal gene transfer in creating novel pathogenicity phenotypes and the likely important relationship between animal and human chlamydial strains. Additionally, the Taxochip design and the Chlamydiales gene database will be of long-term benefit to all chlamydiologists and molecular evolutionists who are increasingly using the Chlamydiales as a standard model for studying reductive evolutionary processes. This study is designed to address broad questions that deal with the genetic and evolutionary basis of the development of intracellular parasitism and pathogenicity in the Chlamydiales. With broad and international support from the scientific community, we consider this a groundbreaking proposal and anticipate it will be a benchmark study in comparative microbial genomics.